1. Field of the Invention
The present invention relates generally to the capacitative entry of calcium ions (Ca.sup.2+) into mammalian cells and the mechanisms by which such capacitative entry is accomplished. More particularly, the present invention is directed to the discovery of transient receptor potential (trp) proteins which are an essential part of the capacitative Ca.sup.2+ entry (CCE) mechanism in mammalian cells. The invention further relates to methods for altering CCE in mammalian cells by controlling the expression of trp proteins or treating the cell with compounds which inhibit the biological activity of the trp protein. The invention also is directed to using the trp proteins as screening agents in methods for identifying compounds which may be useful in controlling CCE in mammalian cells.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and identified in the appended bibliography. The bibliography also includes a number of references which are not specifically referred to in the description. These references are listed as providing additional description of related art.
Calcium regulation plays an important role in many cellular processes. In non-excitable mammalian cells, activation of phosphoinositide-specific phospholipase C (PLC) produces inositol 1,4,5-trisphosphate (IP.sub.3), which in turn causes the release of intracellular calcium from its storage pools in the endoplasmic reticulum. This results in a transient elevation of cytosolic free Ca.sup.2+, which is normally followed by a Ca.sup.2+ influx from the extracellular space. By refilling the pools, Ca.sup.2+ influx plays an important role in prolonging the Ca.sup.2+ signal, allowing for localized signaling, and maintaining Ca.sup.2+ oscillations 1!.
Calcium influx in non-excitable cells is thought to occur through plasma membrane channels which, in contrast to the voltage-dependent Ca.sup.2+ channels in excitable cells, are operated not by changes of membrane potentials but rather by how full the internal Ca.sup.2+ stores are 2!. The Ca.sup.2+ channels have variously been referred to as calcium release-activated calcium channels (CRACs), store-operated calcium channels (SOCs), and receptor-operated calcium channels (ROCs) (23, 24, 25 and 26). Because the entering Ca.sup.2+ replenishes Ca.sup.2+ stores that act like capacitors, it is also called capacitative Ca.sup.2+ entry or CCE (27, 28).
Although studies using either fluorescent Ca.sup.2+ indicators or electrophysiological techniques have suggested that multiple types of Ca.sup.2+ permeant channels may be involved in different cell types to fulfill the influx function, the molecular structure of the channels and the mechanism that regulates the influx have remained unclear and represent one of the major unanswered questions of cellular Ca.sup.2+ homeostasis 3-5!.
Candidates involved in voltage independent Ca.sup.2+ entry into cells include a gene product missing in a Drosophila mutant, the transient receptor potential (trp), and its homologue, trp-like (trp1). The insect phototransduction pathway is mediated through the activation of PLC coupled by a G.sub.q type protein 6!. The consequent generation of IP.sub.3 and the release of Ca.sup.2+ from its intracellular storage pools is believed to lead to the opening of a light sensitive ion channel and generation of a depolarizing receptor potential. Similar to intracellular Ca.sup.2+ changes in mammalian cells following stimulation by agonists acting via PLC, electroretinograms of Drosophila eyes are biphasic with an initial peak followed by a sustained phase of which the latter is dependent on extracellular Ca.sup.2+. This sustained phase is absent in the trp mutant which was therefore proposed to be caused by a defect in the Ca.sup.2+ influx pathway 6!. The trp gene was cloned 7,8!. Subsequently, molecular cloning of a Drosophila calmodulin binding protein showed it to be a homologue of the trp gene product and named trp-like or trp1 9!. A detailed analysis of the trp1 sequence showed that it shares moderate homology with voltage-dependent Ca.sup.2+ and Na.sup.+ channels at their putative transmembrane regions. However, in clear contrast with the voltage-dependent channels, it lacks the positively charged amino acid residues at the presumed S4 segment which are thought to act as voltage sensors that promote gating in response to changes in membrane potentials. The structural homology to Ca.sup.2+ and Na.sup.+ channels together with the absence of charged residues in trp1 and trp suggested that these proteins may form voltage independent ion channels. This was demonstrated recently by expression of the cDNAs for trp and trp1 in insect Sf9 cells using the baculovirus system. It was found that trp forms a Ca.sup.2+ permeable cation channel which is activated by store depletion with thapsigargin 10! whereas trp1 forms a Ca.sup.2+ permeable non-selective cation channel which is not only constitutively active when over-expressed in Sf9 cells but also can be up-regulated by receptor stimulation 11-13!. However, it was also noticed that neither trp nor trp1 mimicked the endogenous Ca.sup.2+ influx channel of the St9 cells, suggesting the existence of at least one other channel in insects involved in Ca.sup.2+ entry 10!.